Comparative analysis of mitochondrial DNA 12S rRNA region between Pelodiscus sinensis and Pelodiscus axenaria and their molecular marker for identification

The primers for Pelodiscus sinensis and Pelodiscus axenaria were designed by using the sequences of mitochondrial DNA 12S rRNA region of Dogania subplana , Chrysemys picta and Chelonia mydas , 562 base pairs of mitochondrial DNA were amplified and sequenced. Theresultsshowthatthelengthoftheirsequenceisthesame,theA,T,G,Ccontentsaresimilar,thenumberofA,T,G, Cis 209 (37.2%) ,121 (21.5%) ,145 (25.8%) ,87 (15.5%)in Pelodiscus sinensis and 207 (36.8%),120 (21.4%),145 (25.8%), 90 (16 %)in Pelodiscus axenaria. Based on their sequence data , we found there are 13 different nucleotide sites (percentage divergence is 2. 31 %) between Pelodiscus sinensis and Pelodiscus axenaria by using the software of ClustalW , whereas the average number of different nucleotide sites is 3 (percentage divergence is 0. 53 %) in Pelodiscus sinensis and 2 (percentage divergence is 0. 36 %) in Pelodiscus axenaria. The nucleotide percentage divergence is obvious between species , so it suggests Pelodiscus axenaria is a new species in Trionyx. Restriction endonuclease analysis based on sequence data of this DNA fragment revealed the presence of polymorphic sites for MspI endonuclease , there is one site for MspI endonuclease in Pelodiscus axenaria , but none in Pelodiscus sinensis. The restriction profiles obtained by agarose gel electrophoresis when amplicons were cut with MspI enzyme allowed the unequivocal identification of Pelodiscus sinensis and Pelodiscus axenaria.